Bottom Line:
We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes.NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene.Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

ABSTRACTDespite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

Figure 3: Quantitative analysis for disassembly and recycling of LNPsa. HeLa cells were exposed to LNP with the FRET pair (AF647/AF594 siRNA), washed and replenished with media. Changes in FRET (excitation-561 nm, emission-641 nm) intensity were monitored at different time points using flow cytometery to quantify LNP disassembly. The emission from a single fluorophore (excitation-633 nm, emission-641 nm) was used to measure intracellular siRNA at these time points. b. Cellular uptake of FRET probe-LNP (siAF647/siAF594, 3hrs) was imaged in stably transfected GFP-tubulin cells 1 hour post incubation c. siAF647-LNP was pulsed for 3 hrs, washed and incubated with fresh media to remove non internalized particles. Media was removed at multiple time points and analyzed using a fluorescent reader to determine amount recycled. 1% Triton-X was later added (red curve) to the media and fluorescence was re-measured. d. Amount of siRNA was obtained from fluorescence values from (c) that were extrapolated using the standard curve (inset).

Mentions:
In addition to the moderate co-localization of LNPs with ERC, our imaging studies also indicated a considerable redistribution of LNPs in the presence of wide variety of compounds from the screen that cause stress on the endoplasmic reticulum. (Supplemental Information Small Molecule Library). Therefore, we wanted to quantify the kinetics of LNP disassembly and the relative contributions of internalization and recycling on siRNA delivery using a FRET probe26. The probe contains two identical siRNA’s, each labelled with a different fluorophore that together form a FRET pair. The siRNA-bound fluorophores aggregate with the cationic lipids in the nanoparticle mixture and undergo FRET, which is indicative of intact LNPs. Flow cytometery data shows a time dependent decrease in FRET based fluorescence signal within 1 hour of internalization, which was indicative of LNP disassembly inside cells (Fig 3a). However, the fluorescence emanating from a single fluorophore (AF647) remained constant during this time. A decline in AF647 signal was observed only after 1-4 hrs which substantially diminished after 24 hrs. Therefore, LNPs disassemble rapidly within the first hour of entry followed by degradation or recycling of siRNA within 24 hrs (Fig 3a). Labelled siRNA was located in vesicular compartments alongside microtubules 1 hour post incubation; indicating that, most siRNA’s remain trapped inside endosomes after nanoparticle disassembly (Fig 3b).

Figure 3: Quantitative analysis for disassembly and recycling of LNPsa. HeLa cells were exposed to LNP with the FRET pair (AF647/AF594 siRNA), washed and replenished with media. Changes in FRET (excitation-561 nm, emission-641 nm) intensity were monitored at different time points using flow cytometery to quantify LNP disassembly. The emission from a single fluorophore (excitation-633 nm, emission-641 nm) was used to measure intracellular siRNA at these time points. b. Cellular uptake of FRET probe-LNP (siAF647/siAF594, 3hrs) was imaged in stably transfected GFP-tubulin cells 1 hour post incubation c. siAF647-LNP was pulsed for 3 hrs, washed and incubated with fresh media to remove non internalized particles. Media was removed at multiple time points and analyzed using a fluorescent reader to determine amount recycled. 1% Triton-X was later added (red curve) to the media and fluorescence was re-measured. d. Amount of siRNA was obtained from fluorescence values from (c) that were extrapolated using the standard curve (inset).

Mentions:
In addition to the moderate co-localization of LNPs with ERC, our imaging studies also indicated a considerable redistribution of LNPs in the presence of wide variety of compounds from the screen that cause stress on the endoplasmic reticulum. (Supplemental Information Small Molecule Library). Therefore, we wanted to quantify the kinetics of LNP disassembly and the relative contributions of internalization and recycling on siRNA delivery using a FRET probe26. The probe contains two identical siRNA’s, each labelled with a different fluorophore that together form a FRET pair. The siRNA-bound fluorophores aggregate with the cationic lipids in the nanoparticle mixture and undergo FRET, which is indicative of intact LNPs. Flow cytometery data shows a time dependent decrease in FRET based fluorescence signal within 1 hour of internalization, which was indicative of LNP disassembly inside cells (Fig 3a). However, the fluorescence emanating from a single fluorophore (AF647) remained constant during this time. A decline in AF647 signal was observed only after 1-4 hrs which substantially diminished after 24 hrs. Therefore, LNPs disassemble rapidly within the first hour of entry followed by degradation or recycling of siRNA within 24 hrs (Fig 3a). Labelled siRNA was located in vesicular compartments alongside microtubules 1 hour post incubation; indicating that, most siRNA’s remain trapped inside endosomes after nanoparticle disassembly (Fig 3b).

Bottom Line:
We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes.NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene.Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

ABSTRACTDespite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.